Rho family GTPases (Rho, Rac, Cdc42) are important intracellular signaling proteins that control diverse cellular functions, including actin cytoskeletal organization, invasion and metastasis, transcriptional regulation, cell cycle progression, apoptosis, vesicle trafficking, and cell-to-cell and cell-to-extracellular matrix adhesions. Consequently, Rho GTPases have been implicated in cancer, and the progression of other diseases by a large number of studies. Of the Rho family GTPases, Rac1 and Rac3, the isoforms expressed in non-hematopoietic cells, have been specifically implicated in rearrangement of the actin cytoskeleton into cell surface protrusions called lamellipodia or invadopodia that are specific for forward migration during invasion; and thus, have been implicated in promotion of metastasis. Racs have also been shown to be essential for Ras and other oncogene-mediated transformation. Our group and others as well, has implicated hyperactive Rac1 and Rac3 with increased survival, proliferation, and invasion of breast and brain cancers. Recent reports have shown a role for Rac in mammalian target of rapamycin (mTOR)-mediated regulation of cancer malignancy and anti breast cancer therapy resistance. Moreover, Rac1 was shown to increase Estrogen receptor-alpha (ERα)-mediated transcriptional activity in breast cancer. Studies have also demonstrated a cancer-promoting role for the constitutively active Rac1b splice variant that is overexpressed in breast and colorectal cancer. Since the malignant phenotype of Rac is associated with activation of its direct downstream effectors p21-activated kinases (PAKs), much effort has been focused on the development of PAK inhibitors as anti cancer therapeutics. However, in addition to PAK, Racs have multiple downstream effectors such as WAVE and Mena/VASP that contribute to cancer. Therefore, targeting Rac is a more viable approach for the development of anticancer drugs.
Unlike the related small GTPase Ras, Racs are not mutated in malignant cancers but rather overexpressed or hyperactivated. Racs are activated by GTP/GDP exchange catalyzed by guanine nucleotide exchange factors (GEF) that are regulated via a myriad of cell surface receptors. So far, over 60 potential Rac-GEFs have been identified. Of these, Dbl family GEFs such as T-cell invasion and metastasis gene product (Tiam-1) and Vav have been implicated in breast cancer progression. Tiam-1 expression has also been correlated with high breast cancer cell migration, indicating a specific role for Tiam-1 in breast cancer metastasis. Recent reports have also shown that PIP3-dependent Rac exchanger 1 (p-Rex1) is upregulated in breast cancer cells and breast cancer patients with poor prognosis. Thus, elevation of Rac.GEF expression and/or activity appears to be a common phenomenon during cancer progression. Therefore, targeting the binding of Rac to GEFs is a rational strategy to inhibit Rac activity and thus, cancer invasion.
NSC23766 was identified as a small molecule that binds to a putative binding pocket in the surface groove of Rac1 that interacts with the Rac-specific GEFs Trio and Tiam1. NSC23766 has been shown to inhibit the anchorage-independent growth and invasion of human prostate cancer PC-3 cells, Rac activation and Rac-dependent aggregation of platelets stimulated by thrombin, Rac1 and Rac2 activities of hematopoietic stem/progenitor cells, and migration from mouse bone marrow to peripheral blood. NSC23766 has also been shown to inhibit invasion of chronic myelogenous leukemia cells in vitro and in vivo in a mouse model. Thus, such structure-function based rational design appears to represent a new avenue for generating small molecule inhibitors of Rac. However, NSC23766 is a moderately active Rac inhibitor with a relatively high IC50 of 50-100 μM in fibroblasts, which limits its potential use as a therapeutic agent. In addition, our group has found that in the highly metastatic cancer cell line MDA-MB-435, NSC23766 inhibits Rac1 by only ˜20% at a concentration of 50 μM, and that at this concentration there is no significant effect on lamellopodia formation. Therefore, there is a need for more effective inhibitors to reduce Rac activity in highly metastatic cancer cells.
The identification of novel inhibitors of Rac that function via different inhibitory mechanisms has been the subject of several studies. Thus, whereas NSC23766 inhibits the interaction of Rac1 with several of its GEFs, the Rac inhibitor EHT 1864 interferes with the interaction of Rac with its downstream effectors at concentrations of 10-50 μM. A virtual screening of a selected subset of compounds from the ZINC database for binding affinity to Rac1 based on the crystal structure of Rac1 with NSC23766, identified several novel Rac1 inhibitors with experimental IC50 values ranging from 12.2-57 μM. In addition, a high-throughput flow-cytometry bead-based multiplex assay identified MLS000532223 as a compound that prevents GTP binding to Rac. However, other Rho GTPases, such as Cdc42, are also affected by this compound. Small molecule compounds have also been synthesized to specifically inhibit the Rac1b splice variant. Another report identified ITX3 as a GEF inhibitor that targeted Rac and RhoG interaction with Trio; however, this compound is effective at high 50-100 mM concentrations.